The N.sup..alpha. -Bpoc group, which was introduced by Sieber and Iselin in 1968 (1) has played a key role in the synthetic strategy used in several precedent-setting solution phase peptide syntheses of historical note, in particular the first total synthesis of human insulin (2,3) and in the total synthesis of an analog of hen egg white lysozyme (4). Bpoc amino acids have also been applied to solid phase peptide synthesis. Merrifield and Mojsov employed Bpoc amino acids in their solid phase synthesis of crystalline glucagon (6), and Kemp and coworkers have used Bpoc amino acids to do solid phase synthesis on a phenyl ester resin, preparing 4-(peptidyloxy)-6-mercaptodibenzofuran esters for use in thiol capture (9, 11, 12) ligations that have resulted in the completed syntheses of peptides of 25, 29, 34, and 39 amino acids in length. Other studies of solid phase synthesis with Bpoc-amino acids have resulted in the preparation of a nonapeptide from bovine pituitary growth hormone (13), a heptapeptide from bovine parathyroid hormone (14) tyrosine containing analogs of the ion-channel forming pentadecapeptide, Gramacidin A (15), and a 42-amino acid sequence corresponding to a highly acidic fragment of nucleolar nonhistone protein C23 (16).
In the work described above, the application of Bpoc amino acids to solid phase peptide synthesis was done by first preparing and storing the cyclohexyl- or dicyclohexylamine salts of each derivative, which then had to be liberated from the salt and activated prior to each coupling. Most of the Bpoc amino acids, as free acids, are obtained as oils which have a relatively short shelf-life. The oils undergo an autocatalytic decomposition to the amino acid, CO.sub.2 and to the Bpoc olefin and its dimer with a half-life of weeks. Bpoc-Leu-OH and Bpoc-Asn-OH, which alone among the free acids, are obtained as crystalline solids, have a longer shelf-life of at least a year if kept dry at -20.degree. C.
Certain active esters of Bpoc amino acids (usually hydroxysuccinimide, nitrophenyl, 2,3,5-trichlorophenyl, or pentachlorophenyl) were prepared and stored for use in the solution phase peptide syntheses listed above, but the esters used were not efficient enough to be practical for application to solid-phase synthesis.
The favorable properties of Pfp esters were noted by Kovacs, who in his study of N.sup..alpha. -urethane-protected cysteine derivatives (20), identified Pfp esters as having the highest k.sub.coup / k.sub.rac ratio of a wide range of active esters studied. Kisfaludy (22) first prepared Pfp esters of Fmoc amino acids that were later applied by Atherton and Sheppard (24) to solid phase synthesis in their Fmoc/polyamide continuous flow system. In a recent comprehensive study, Hudson (26) has reported simultaneous competition and comparison methods to evaluate the activities of twenty-six reactive esters of N.sup..alpha. -Fmoc protected amino acids. Pfp esters were identified as being the most suitable selection for routine use in solid-phase synthesis.
The in situ preparation of 3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl (ODhbt) esters of certain urethane protected amino acids was disclosed by Konig and Geiger (43), but did not gain favor due to the formation of an o-azidobenzoic acid ester as a by-product of their preparation. Atherton and Sheppard (24) demonstrated the usefulness of pre-formed ODhbt esters of Fmoc-amino acids, if they were first crystallized and obtained in a homogeneous state free of the azido impurity formed in the in situ preparation.
A recent review of solid-phase peptide synthesis is provided in Barany, G. et al.(47) This reference is specifically incorporated by reference in its entirety herein to provide details of solid-phase synthetic methods.
The present work relates to Pfp and ODhbt activated esters of N.sup..alpha. -Bpoc amino acids, particularly those that are crystalline, which have apparently not been reported previously.